All membranes of living cells contain glycerophospholipids which have fatty acid attached to the 1 and 2 carbons of the 3-carbon glycerol molecule. The fatty acids at these two positions have different numbers of carbon atoms and degrees of unsaturation (a double bond between the two carbon atoms). The length of the fatty acid and degree of saturation have important dietary consequences for man. For example, diets rich in saturated fatty acids are associated with increased risk of coronary artery disease whereas monounsaturated fatty acids are associated with decreased risk. Plant seed also consist largely of triacylglycerol-glycerol having three fatty acids.
The type of fatty acids present in glycerolipids is determined by enzymes called fatty acyltransferases. The present inventors isolate the first eucaryotic gene, SLC1 that encodes a transferase specific for the 2 position of glycerolipids from Saccharomyces cerevisiae.
Determining the biological function(s) and mode of action of individual sphingolipids in multicellular eucaryotes has proven to be a challenging and unfinished task because of the great variety of sphingolipids present in such organisms (1-3, see reference citations below). In contrast to this variety, the unicellular eucaryote Saccharomyces cerevisiae has only one major and two minor types of structurally related sphingolipids. Such simplicity provides a unique opportunity to study sphingolipid function(s) in an organism to which molecular genetic techniques can be readily applied.
The most abundant sphingolipid in S. cerevisiae is mannosyldiinositolphosphorylceramide with lesser amounts of inositolphosphorylceramide and mannosylinositolphosphorylceramide (4,5). The ceramide moiety contains the sphingoid long chain base phytosphingosine linked by an amide bond to a C.sub.26 fatty acid (4).
Synthesis of phytosphingosine initiates with the condensation of serine and palmitoyl-CoA catalyzed by serine palmitoyltransferase to yield 3-ketodihydrosphingosine (6). In S. cerevisiae the enzyme or one of its subunits is encoded by the LCB1 gene (7). Strains defective in lcb1 lack serine palmitoyltransferase activity and have an Lcb.sup.- phenotype because they require a long chain base such as phytosphingosine for growth (8-10). The Lcb.sup.- phenotype provides a starting point for a molecular genetic analysis of sphingolipid function(s). Saccharomyces cerevisiae normally requires sphingolipid biosynthesis for growth.
Some glycerolipids of Saccharomyces cerevisiae are known. For example, U.S. Pat. No. 5,057,419 is entitled Genetically Engineered Plasmid and Organisms for the production of Specialized Oils. This patent discloses an expression vector encoding a yeast delta-9 fatty acid desaturase enzyme which functions in a yeast cell to induce or enhance oil production. The overproduction of delta-9 desaturase by the cells leads to the production of abnormally high levels of unsaturated fatty acids in the cell membrane. In order to compensate for the increased levels of unsaturated fatty acids in the lipids, excess unsaturated fatty acids are removed from the membrane lipids and shunted into triglyceride formation. The yeast are indicated to overproduce oils containing polyunsaturated fatty acid with superior properties.
U.S. Pat. No. 5,288,619 to Moore discloses a process for industrial enzymatic interesterification of a triglyceride including steps of reacting a triglyceride in an enzyme conversion zone. The enzymes which are used are preferably 1,3 specific lipases.
U.S. Pat. No. 5,286,633 to Brown et al. discloses an enzymatic transesterification method for preparing a margarine oil. The margarine oil product has a non-random fatty acid distribution in which esterified stearic acid is predominantly distributed in the 1,3-positions while esterified unsaturated fatty acid moieties are in higher concentration in the 2- position of the glycerides. The method of making the margarine oil includes a step of providing a transesterification reaction mixture comprising stearic acid and triglyceride vegetable oil and transesterifying using a 1,3 lipase. Enzymes from synthetic sources are contemplated, including yeasts.
Chemical Abstracts, Vol. 92, Abstract 17761w, (1980) "Characterization of sterol ester synthetase in Saccharomyces cerevisiae" discloses that cell free extracts of Saccharomyces cerevisiae catalyzed the synthesis of fatty acid ester of sterol from cholesterol, fatty acid, ATP, and CoA or from cholesterol and fatty acyl CoA. The enzyme involved in the formation of the ester is acyl-CoA-sterol-O-acyltransferase.
Chemical Abstracts, Vol. 91, Abstract 188621j, (1979) "Utilization of endogenous diacyl glycerol for the synthesis of triacylglycerol, phosphatidylcholine and phosphatidyl ethanolamine by lipid particles from baker's yeast" discloses a measurement of the activity of 3 enzymes prepared from S. cerevisiae in the presence of 1,2-diacylglycerol substrates. The enzymes include diacylglycerol acyltransferase, choline phosphotransferase, and ethanolamine phosphotransferase.
Chemical Abstracts, Vol. 88, Abstract 165910b, (1978), "Glycerolipid biosynthesis in Saccharomyces Cerevisiae" discloses an investigation of S. cerevisiae dihydroxyacetone phosphate acyltransferase to determine whether its activity and that of glycerol phosphate acyltransferase represent dual catalytic functions of a single membranous enzyme.
Chemical Abstracts, Vol. 87, Abstract 129342p, (1977), "Acyltransferase systems involved in phospholipid metabolism in Saccharomyces cerevisiae" discloses membrane preparations of S. cerevisiae catalyzed the acylation of glycerophosphate, 1 acyl- and 2 acyl-glycerophosphates and 1 acyl- and 2 acyl-glycerylphosphocholines. Specificity of glycerophosphate acyltransferase, 2-acylglycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase were determined.
Chemical Abstracts, Vol. 105, Abstract 94333x, (1986), "Mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase: Simultaneous loss of dihydroxy acetone phosphate acyltransferase indicates a common gene" discloses the isolation of fourteen independent mutants defective in sn-glycerol-3-phosphate acyltransferase activity.
Chemical Abstracts, Vol. 115, Abstract 274626k, (1991), "Acyl CoA-cholesterol acyltransferase (ACAT) inhibitors from microbial and plant lipids" discloses that enzyme ACAT, responsible for absorption of cholesterol by the intestinal epithelium is inhibited by fatty acid-like substances obtained from enzymatic degradation products of microbial and plant products.
Chemical Abstracts, Vol. 114, Abstract 38917q, (1991), is directed to "Genetic and biochemical studies of sn-glycerol-3-phosphate acyltransferase in Saccharomyces cerevisiae". Chemical Abstracts, Vol. 117, Abstract 167433q, (1992), "The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae" discloses studies of the acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis. This pathway is used in yeast Saccharomyces cerevisiae non-ether glycerolipid synthesis.
There is a need in the art for a knowledge of the SLC1 gene of Saccharomyces cerevisiae and its use to construct economically and dietetically important plants, such as seeds, from which cooking oils are obtained which have fatty acids with optimal benefits, as well as better storage properties. Likewise, there is a need to produce animals whose meat products would contain glycerolipids with fatty acids having different degrees of saturation and/or chain length. Such changes improve the flavor and storage properties and reduce adverse effects on humans, for example, by reducing the percentage of polyunsaturated fatty acids. The present invention provides these benefits and overcomes the deficiencies and lack of knowledge of the prior art.